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. 2017 Aug;66(8):1414-1427.
doi: 10.1136/gutjnl-2016-313099. Epub 2017 Mar 21.

Dietary Emulsifiers Directly Alter Human Microbiota Composition and Gene Expression Ex Vivo Potentiating Intestinal Inflammation

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Dietary Emulsifiers Directly Alter Human Microbiota Composition and Gene Expression Ex Vivo Potentiating Intestinal Inflammation

Benoit Chassaing et al. Gut. .
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Abstract

Objective: The intestinal microbiota plays a central role in the development of many chronic inflammatory diseases including IBD and metabolic syndrome. Administration of substances that alter microbiota composition, including the synthetic dietary emulsifiers polysorbate 80 (P80) and carboxymethylcellulose (CMC), can promote such inflammatory disorders. However, that inflammation itself impacts microbiota composition has obfuscated defining the extent to which these compounds or other substances act directly upon the microbiota versus acting on host parameters that promote inflammation, which subsequently reshapes the microbiota.

Design: We examined the direct impact of CMC and P80 on the microbiota using the mucosal simulator of the human intestinal microbial ecosystem (M-SHIME) model that maintains a complex stable human microbiota in the absence of a live host.

Results: This approach revealed that both P80 and CMC acted directly upon human microbiota to increase its proinflammatory potential, as revealed by increased levels of bioactive flagellin. The CMC-induced increase in flagellin was rapid (1 day) and driven by altered microbiota gene expression. In contrast, the P80-induced flagellin increase occurred more slowly and was closely associated with altered species composition. Transfer of both emulsifier-treated M-SHIME microbiotas to germ-free recipient mice recapitulated many of the host and microbial alterations observed in mice directly treated with emulsifiers.

Conclusions: These results demonstrate a novel paradigm of deconstructing host-microbiota interactions and indicate that the microbiota can be directly impacted by these commonly used food additives, in a manner that subsequently drives intestinal inflammation.

Keywords: INFLAMMATION; INTESTINAL MICROBIOLOGY.

Conflict of interest statement

Competing interests: None declared.

Figures

Figure 1
Figure 1. CMC and P80 do not impact intestinal microbiota composition in ASF mice
Six-week-old ASF C57BL/6 mice were exposed to CMC or P80 diluted in drinking water (1.0%) for 11 weeks. (A–I) Total fecal DNA was extracted and subjected to q-PCR using specific primers for 16sRNA (A), C. sp. (ASF 356) (B), L. intestinalis (C), L. murinus (D), M. schaedleri (E), E. plexicaudatum (F), Firmicutes bacterium (G), C. sp. (ASF 502) (H) and P. sp. (I). Results are expressed as bacteria number per mg of stool (A), using a standard curve, or as relative values (B–I). (J–M) Principal coordinate analysis was performed using euclidean distance with the 8 ASF members at week 6 (J), week 8 (K), week 10 (L) and week 17 (M). Data are the means +/− S.E.M (N=3–6). For clustering analyzing on principal coordinate plots, categories were compared and statistical significance of clustering were determined using Permanova method.
Figure 2
Figure 2. CMC and P80 do not induce intestinal inflammation nor metabolic syndrome nor microbiota encroachment in ASF mice
Six-week-old ASF C57BL/6 mice were exposed to CMC or P80 diluted in drinking water (1.0%) for 11 weeks. (A) Final body weight, (B) fat pad weight, (C) 5hr fasting blood glucose concentration, (D) spleen weight, (E) colon weight, (F) colon length, (G) ceacum weight, (H) liver weight, (I) fecal Lcn2, (J) LPS and (K) FliC levels at 4 weeks and 12 weeks. (L) Confocal microscopy analysis of microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (Blue). Bar=20μm. (M) Distances of the closest bacteria to intestinal epithelial cells (IEC) per condition over 3 high-powered fields per mouse. Data are the means +/− S.E.M (N=3–6). Each treatment group was compared with the control group (water-treated) and statistical significance was determined using Student’s t test.
Figure 3
Figure 3. P80 alters M-SHIME microbiota composition
A 9-vessels mucosal-M-SHIME system was set up and inoculated with fresh human feces at day −7. Following a 7-day stabilization period, vessels were treated with either water (N=2), CMC 1% (w/v, N=3) or P80 1% (v/v, N=3) for 13 days. Luminal microbiota composition was analyzed using Illumina sequencing of the V4 region of 16S rRNA genes. (A–C) Principal coordinates analysis (PCoA) of the weighted UniFrac distance matrix at days −6 and 11 (A), day −6 (B) and day 11 (C). (D–E) Average of the weighted UniFrac distance within group (water, CMC and P80) and between group (Water vs CMC and Water vs P80) has been calculated at days −6 (D) and 11 (E). Data are the means +/− S.E.M (N=2–3). For clustering analyzing on principal coordinate plots, categories were compared and statistical significance of clustering were determined using the Permanova method.
Figure 4
Figure 4. Both CMC and P80 alter microbial gene expression in the mucosal-M-SHIME model
A 9-vessel mucosal-M-SHIME system was set up and inoculated with fresh human feces at day −7. Following a 7-day stabilization period, vessels were treated with either water (N=2), CMC 1% (w/v, N=3) or P80 1% (v/v, N=3) for 13 days. M-SHIME-associated luminal metatranscriptome was analyzed by Illumina sequencing. (A–B) Following functional classification of the reads to Kyoto Encyclopedia of genes and genomes (KEGG) orthology classification at level 4 using MG-RAST, principal coordinates analysis (PCoA) of the euclidean distance matrix were ploted at days −6, 1 and 11. (C) Average of the euclidean distance within group (water) and between group (Water vs CMC and Water vs P80) at days −6, 1 and 11. (D) KEGG orthology at level 4 were visualized on a volcano plot. Up/left: water-treated versus CMC-treated at day1; up/right: water-treated versus P80-treated at day1; bottom/left: water-treated versus CMC-treated at day11; bottom/left: water-treated versus P80-treated at day11. For each KEGG identifier level 4, the difference in abundance between the two groups is indicated in log2 fold change on x-axis (with positive values corresponding to an increase in emulsifier-treated group compare to water-treated group, and negative values corresponding to a decrease in emulsifier-treated group compare to water-treated group), and significance between the two groups is indicated by −log10 p-value on the y-axis. Red dots correspond to KEGG identifiers with a p-value <0.05 between emulsifier-treated and water-treated groups. Orange dots correspond to KEGG identifiers with at least a 2-fold decreased or increased abundance in emulsifier-treated group compare to water-treated group. Green dots correspond to KEGG identifiers with at least a 2-fold decreased or increased abundance in emulsifier-treated group compare to water-treated group and with a p-value <0.05. (E) Heatmap representation using Gene-E of pathways related to flagella synthesis identified using KEGG orthology (KO), Clusters of Orthologous Groups of proteins (COG) and Non-supervised Orthologous Groups (NOG). Data are the means +/− S.E.M (N=2–3).
Figure 5
Figure 5. CMC and P80 increase the pro-inflammatory potential of the mucosal-M-SHIME system microbiota
A 9-vessel mucosal-M-SHIME system was set up and inoculated with fresh human feces at day −7. Following a 7-day stabilization period, vessels were treated with either water (N=2), CMC 1% (w/v, N=3) or P80 1% (v/v, N=3) for 13 days. (A) Luminal M-SHIME suspension FliC levels at days −7, −6, −3, −2, 1, 4, 6, 8, 11 and 13 days. (B) Luminal M-SHIME suspension was injected intraperitoneally to Rag−/− mice, and IL-6 were measured in the serum 2 h post-injection. Data are the means +/− S.E.M (N=3). Each treatment group was compared with the control group (water-treated) and statistical significance was determined using Student’s t test.
Figure 6
Figure 6. CMC and P80 increase the pro-inflammatory potential of the mucosal-M-SHIME system microbiota
A 9-vessel mucosal-M-SHIME system was set up and inoculated with fresh human feces at day −11. Following an 11-day stabilization period, vessels were treated with either water, CMC (0.10%, 0.25%, 0.50% or 1.00%) or P80 (0.10%, 0.25%, 0.50% or 1.00%) for 10 days. (A–B) Luminal M-SHIME suspension LPS (A) and FliC (B) levels of CMC-treated M-SHIME at days −10, −7, −4, −2, 0, 1, 2, 3, 5, 7 and 10 days. (C–D) Luminal M-SHIME LPS (C) and FliC (D) levels of CMC- and P-80-treated M-SHIME at days −10, −7, −4, −2, 0, 1, 2, 3, 5, 7 and 10 days. Data are expressed as relative values with day −1 defined as 1 (N=1).
Figure 7
Figure 7. Altered microbiota composition in mice receiving CMC- and P80-treated M-SHIME suspensions
A 9-vessel M-SHIME system was set up and inoculated with fresh human feces at day −7. Following a 7-day stabilization period, vessels were treated with either water, CMC 1% or P80 1% for 13 days. Germfree C57BL/6 mice (4 weeks old) were removed from isolator and were orally administered with 200μL of luminal M-SHIME suspension (day 11 sample). Transplanted mice were then housed in isolated ventilated cages. For each condition (water-treated M-SHIME, CMC-treated M-SHIME, P80-treated M-SHIME), 2 cages containing 2–3 animals were used, with each cage receiving suspension from an independent M-SHIME vessel (for each condition, N=2 vessel, N=2 cages, and N=5 animals). Fecal microbiota composition was analyzed using Illumina sequencing of the V4 region of 16S rRNA genes. (A) Microbiota composition was analyzed from GSU conventional mice, luminal M-SHIME suspension and germfree mice colonized with luminal M-SHIME suspension using Illumina sequencing of the V4 region of 16S rRNA genes. (B–C) Principal coordinates analysis (PCoA) of the unweighted UniFrac distance matrix at days 13 (B) and 64 (C). Samples were colored by treatment (left side) or by cage (right side) (N=5). For clustering analyzing on principal coordinate plots, categories were compared and statistical significance of clustering were determined using the Permanova method.
Figure 8
Figure 8. CMC- and P80-treated M-SHIME suspensions promote low-grade inflammation when transplanted to germfree recipient mice
A 9-vessel mucosal-M-SHIME system was set up and inoculated with fresh human feces at day −7. Following a 7-day stabilization period, vessels were treated with either water, CMC 1% or P80 1% for 13 days. Germfree C57BL/6 mice (4 weeks old) were removed from isolator and were orally administered with 200μL of luminal M-SHIME suspension (day 11 sample). Transplanted mice were then housed in isolated ventilated cages. For each condition (water-, CMC-, and P80-treated M-SHIME), 2 cages containing 2–3 animals were used, with each cage receiving suspension from an independent M-SHIME vessel (for each condition, N=2 vessel, N=2 cages, and N=5 animals). (A, C) LPS and (B, D) FliC levels at days 13 (A, B) and 64 (C, D). (E) Body weight over time, (F) 15hr fasting blood glucose concentration, (G) 5hr fasting blood glucose concentration, (H) fat pad weight, (I) colon weight, (J) colon length, (K) colon weight/length ratio, (L) spleen weight and (M–N) fecal Lcn2 levels at days 13 (M) and 64 (N). Data are the means +/− S.E.M (N=5). Each treatment group was compared with the control group (water-treated) and statistical significance was determined using Student’s t test.
Figure 9
Figure 9. CMC- and P80-treated M-SHIME suspensions promote microbiota encroachment when transplanted to germfree recipient mice
A 9-vessel mucosal-M-SHIME system was set up and inoculated with fresh human feces at day −7. Following a 7-day stabilization period, vessels were treated with either water, CMC 1% or P80 1% for 13 days. Germfree C57BL/6 mice (4 weeks old) were removed from isolator and were orally administered with 200μL of luminal M-SHIME suspension (day 11 sample). Transplanted mice were then housed in isolated ventilated cages Isocages (Techniplast). (A) Confocal microscopy analysis of microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (Blue). Bar=20μm. (B) Distances of the closest bacteria to intestinal epithelial cells (IEC) per condition over 3 high-powered fields per mouse. Data are the means +/− S.E.M (N=5). Each treatment group was compared with the control group (water-treated) and statistical significance was determined using Student’s t test.

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